Influence of mRNA Turnover on the Biology of Cellular Senescence and Aging

National Institute On Aging

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Abstract

Changes in gene expression patterns are a hallmark of the aging process. Important insight into the mechanisms controlling such gene expression programs has come from the study of replicative senescence of cultured cells (eg, human diploid fibroblasts), which recapitulates many facets of cells from aging individuals. This Project has traditionally studied changes in RBP expression and function during replicative senescence. It has also examined the influence of RBPs in replicative senescence by interventions to elevate or reduce RBP levels, followed by the analysis of changes in senescence-associated mRNA expression patterns. We study if a given RBP binds a senescence-associated mRNA using a variety of in vitro binding assays (biotin pulldown, RNA EMSA, surface plasmon resonance/Biacore, etc) and assays to measure binding of endogenous molecules (ribonucleoprotein immunoprecipitation). To investigate RBP function during senescence, we employ approaches such as RBP silencing, RBP overexpression, and the identification of RBP-associated mRNAs using microarrays. We investigate whether RBPs affect the stability of target mRNAs during senescence, we measuring the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs and use various senescence-associated markers to examine changes in the senescence phenotype.[unreadable] [unreadable] During the past funding period, we have expanded on our recent studies that identified SIRT1 (sirtuin 1) mRNA as a target transcript of the RBP HuR. As HuR levels decline with replicative senescence, so does the levels and stability of SIRT1 mRNA and consequently the SIRT1 protein and the levels of numerous other HuR target mRNAs.[unreadable] [unreadable] Ongoing studies are examining the consequences of the senescence-associated reduction in levels of RBPs AUF1, HuR, TIAR, TIA-1, NF90, and Nucleolin. In one particular study, we have identified that the stability of the heme oxygenase-1 (HO-1) mRNA is regulated by HuR in human diploid fibroblasts (IMR-90). As the project expands into newer areas, we are systematically assessing the levels of RBPs in different mouse tissues throughout the animal life span using tissue microarrays. In humans, we are testing RBP levels in individuals of increasing ages, starting with the intestinal epithelium.